Immunoglobulin G (IgG) Antibody Overview

What is IgG Antibody

IgG is a central element of the adaptive immune system, renowned for its crucial role in the body's defense mechanisms. As the most prevalent antibody class in human serum, IgG constitutes approximately 75% of the total antibodies circulating in the bloodstream. Its prominence underscores its significance in both innate and acquired immunity. IgG antibodies are instrumental in identifying and neutralizing a diverse array of pathogens, including bacteria, viruses, and fungi. They achieve this by specifically binding to antigens—foreign substances that provoke an immune response—thereby facilitating their removal from the body. This binding not only neutralizes the pathogen directly but also enhances the overall immune response through various mechanisms.

Structure of IgG

General Structure

IgG antibodies are tetrameric proteins with a molecular weight of approximately 150 kDa. They are composed of four polypeptide chains: two identical heavy chains and two identical light chains. The structure of IgG can be described as a Y-shaped molecule with a flexible hinge region that connects the arms of the Y.

Domains of IgG

Fab Region: Each IgG molecule has two antigen-binding fragments (Fab regions) at the tips of the Y. These regions contain variable domains (VL and VH) that bind specifically to antigens.

Fc Region: The trunk of the Y-shaped antibody is the Fc region, composed of constant domains (CH2 and CH3) of the heavy chains. This region interacts with Fc receptors on immune cells and activates the complement system.

Subclasses of IgG

IgG is divided into four subclasses, each with distinct properties and functions:

IgG1 is the most prevalent IgG subclass, making up about 65% of total IgG. It is highly effective in neutralizing toxins, viruses, and activating the classical complement pathway, enhancing immune responses through phagocytosis and antibody-dependent cellular cytotoxicity (ADCC).

IgG2 accounts for about 25% of total IgG and is essential for defense against encapsulated bacterial pathogens. Although less effective in complement activation, it is crucial for responding to polysaccharide antigens.

IgG3 makes up approximately 8% of total IgG and is known for its strong complement activation capabilities. It plays a significant role in immune responses and pathogen clearance through enhanced Fc receptor binding.

IgG4, comprising about 5% of total IgG, is unique due to its ability to swap antigen-binding sites with other IgG4 molecules, reducing inflammatory responses. It is associated with tolerance to allergens and is often elevated in allergic conditions and IgG4-related disease.

Functions of IgG

Mechanisms of Action

Neutralization: IgG antibodies neutralize pathogens by binding to specific antigens on their surface. This binding prevents the pathogens from entering host cells and initiating infection.

Opsonization: IgG tags pathogens for phagocytosis by immune cells. The Fc region of IgG binds to Fc receptors on phagocytes, enhancing the uptake and destruction of the pathogens.

Complement Activation: IgG activates the classical complement pathway. This activation leads to the formation of the membrane attack complex (MAC), which lyses pathogen cells and promotes their clearance.

Antibody-Dependent Cellular Cytotoxicity (ADCC): IgG-coated cells are recognized and destroyed by immune cells, such as natural killer (NK) cells. This process involves the binding of IgG to Fc receptors on NK cells, leading to targeted cell lysis.

Transplacental Transfer: IgG is the only antibody class capable of crossing the placenta. This transfer provides passive immunity to the fetus, protecting it from infections during early life.

Clinical Significance

IgG levels can provide insights into an individual's immune status and history of exposure to specific pathogens. Elevated or reduced IgG levels can indicate immune disorders, chronic infections, or other health conditions.

Role in Inflammation: IgG antibodies are integral to inflammatory responses. They bind to antigens, facilitating their recognition and elimination by immune cells. This process can lead to the activation of various inflammatory pathways, which are essential for combating infections. However, chronic or dysregulated IgG responses can contribute to persistent inflammation and tissue damage. Conditions such as rheumatoid arthritis and lupus are examples where abnormal IgG-mediated inflammation plays a role in disease pathology.

Support to the Immune System: IgG antibodies are fundamental to the immune system's function. They neutralize pathogens, preventing their entry into host cells and tissues. By binding to antigens, IgG enhances phagocytosis through opsonization, where immune cells more effectively engulf and destroy pathogens. Additionally, IgG activates the complement system, a cascade of proteins that further aids in the destruction of pathogens and the clearance of immune complexes.

What is the difference between IgA and IgG and IgM

Clinical Relevance of IgG Antibodies

Cytomegalovirus (CMV) IgG Antibody

CMV IgG antibodies signify past exposure to the cytomegalovirus, a common herpesvirus that can cause significant health issues, particularly in immunocompromised individuals and newborns. The presence of CMV IgG is used to confirm a previous infection, as these antibodies typically remain in the body for life once produced. Testing for CMV IgG is crucial for diagnosing the history of CMV infection in individuals who might be experiencing symptoms consistent with CMV or for those who are at risk of congenital CMV infection. In pregnant women, the detection of CMV IgG can help assess the risk of passing the virus to the fetus, which can lead to severe developmental issues and other complications in newborns.

CCP Antibodies (IgG/IgA)

Anti-cyclic citrullinated peptide (CCP) antibodies, including both IgG and IgA subclasses, are significant biomarkers for rheumatoid arthritis (RA). These antibodies target citrullinated proteins, which are modified forms of normal proteins implicated in the pathogenesis of RA. The presence of CCP antibodies is strongly associated with RA, and their levels can reflect disease activity and progression. Elevated levels of CCP antibodies are often observed in patients with RA, and these antibodies can help differentiate RA from other types of arthritis. Additionally, CCP antibody testing can be used to predict disease onset in at-risk individuals and to monitor the effectiveness of therapeutic interventions.

Rubella IgG Antibody

Rubella IgG antibodies are indicative of either past infection with the rubella virus or successful vaccination against rubella, a contagious viral disease that can cause serious birth defects if contracted during pregnancy. High levels of Rubella IgG are typically found in individuals who have been vaccinated or previously infected, providing immunity against future infections. This antibody test is essential for assessing vaccination status, especially in pregnant women, to ensure they are protected against rubella and to prevent congenital rubella syndrome, which can lead to severe birth defects in newborns. Monitoring Rubella IgG levels helps in managing public health strategies and ensuring adequate immunity in vulnerable populations.

IgG Antibodies Sequencing Methods

Mass Spectrometry-Based Antibody Sequencing

Mass spectrometry based antibody sequencing involves analyzing the protein products directly, offering a detailed characterization of IgG antibodies. This method is especially valuable for its ability to bypass the need for antibody-producing cells and directly target polypeptides. This method excels in providing detailed sequence information directly from protein products, making it suitable for complex antibody mixtures and scenarios where antibody-producing cells are not available.

Antibody de novo sequencing involves the digestion of IgG antibodies into smaller peptide fragments, which are then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The peptides' mass-to-charge ratios are determined, and bioinformatics tools reconstruct the entire amino acid sequence. De novo sequencing is particularly advantageous for identifying novel antibodies or analyzing antibodies when no reference sequence is available. It is capable of handling complex mixtures and provides detailed sequence information, including post-translational modifications.

PCR-Based Antibody Sequencing

PCR-based antibody sequencing of IgG antibodies involves the amplification of the variable regions of the antibody genes from the mRNA of antibody-producing cells. This approach is cost-effective and accessible, ideal for generating specific IgG sequences from antibody-producing cells, and is well-suited for recombinant antibody production.

Phage immunoprecipitation sequencing (PhIP-Seq) Antibody Sequencing

PhIP-Seq is a high-throughput method that combines phage display technology with next-generation sequencing to analyze antibody repertoires. PhIP-Seq is suitable for IgG sequencing due to its ability to handle large-scale and multiplexed antibody analyses. It allows for the identification of pathogen-specific antibodies and detailed mapping of antibody repertoires, making it a powerful tool for studying immune responses and vaccine efficacy.

Personalized Medicine

Tailored Therapies: Analyzing an individual's IgG profile can guide the customization of therapeutic interventions, particularly in immunotherapy and allergy treatment.

Predictive Biomarkers: Identifying IgG biomarkers for disease predisposition, prognosis, and therapeutic response helps in developing personalized treatment plans

IgG Purification Methods

The purification of IgG antibodies is a critical step in the biopharmaceutical production process. The goal is to obtain antibodies with high purity and activity, suitable for therapeutic applications. Several purification methods are employed, often in combination, to achieve this:

Protein A/G Affinity Chromatography: This is the most common method for IgG purification. Protein A or G ligands are immobilized on a chromatography matrix and selectively bind the Fc region of IgG antibodies. This allows for the efficient capture and subsequent elution of the antibodies with high purity.

Ion Exchange Chromatography: This technique separates proteins based on their charge. By adjusting the pH and ionic strength of the buffer, IgG antibodies can be selectively bound to and eluted from the chromatography resin. Ion exchange chromatography is often used to remove charged impurities.

Hydrophobic Interaction Chromatography (HIC): HIC separates proteins based on their hydrophobicity. Antibodies are bound to the hydrophobic matrix at high salt concentrations and eluted by decreasing the salt concentration. HIC is useful for removing hydrophobic impurities and aggregates.

Size Exclusion Chromatography (SEC): Also known as gel filtration, SEC separates proteins based on their size. It is used to remove aggregates and ensure the monodispersity of the IgG antibody product. SEC is typically used as a final polishing step.

Multimodal Chromatography: This method combines different modes of interaction (e.g., ion exchange, hydrophobic) in a single chromatography resin, providing enhanced selectivity and purification efficiency. Multimodal chromatography can streamline the purification process by reducing the number of required steps.

Protein L Chromatography: Protein L binds to the kappa light chain of IgG antibodies and can be used to purify antibodies that do not bind well to Protein A or G. This method is particularly useful for certain subclasses of IgG and for antibodies that have been engineered with specific light chain modifications.

Post-translational Structural Modifications of Ig G

IgG undergoes various post-translational modifications that are pivotal in determining its stability, functionality, and therapeutic efficacy. These modifications can significantly impact the antibody's performance in clinical and research applications.

Glycosylation: Glycosylation refers to the covalent attachment of carbohydrate chains to the polypeptide backbone of IgG. By shielding the antibody from proteolytic degradation and oxidative damage, the carbohydrate chains enhance IgG stability, thereby prolonging its half-life in circulation. Additionally, glycan structures modulate IgG's binding affinity to Fc receptors on immune cells and influence complement activation. Variations in glycosylation can affect the efficacy of ADCC and alter the overall immune response. 

Oligomerization: Oligomerization involves the association of multiple IgG molecules through non-covalent interactions, forming complexes such as dimers, trimers, or higher-order aggregates. While monomeric IgG typically retains effective antigen-binding properties, oligomeric forms may exhibit altered binding affinities or diminished functional activity. Additionally, oligomerization can affect how IgG interacts with immune cells and the complement system, influencing its ability to form immune complexes and participate in processes such as opsonization and complement-mediated lysis.

Cleavage and Processing: Cleavage and processing of IgG involve the enzymatic breakdown of the antibody molecule into smaller fragments. This fragmentation yields Fab and Fc fragments, each with distinct functional properties. Fab fragments retain the antigen-binding capability but lack the Fc region's ability to activate immune responses. In contrast, Fc fragments can interact with Fc receptors on immune cells, influencing their activation and function. Controlled cleavage of IgG is strategically employed in therapeutic and diagnostic applications, such as generating Fab fragments for use when full-length antibodies may be too large or cause adverse effects.

Applications of IgG Analysis

Biopharmaceutical Development

Monoclonal Antibodies (mAbs): Ensuring the quality, purity, and potency of mAbs, which are predominantly of the IgG class, requires rigorous analysis throughout the production process.

Biosimilars: Comparing IgG biosimilars to original biologics involves detailed structural and functional analysis to ensure they are equivalent in efficacy and safety.

Disease Diagnosis

Infectious Diseases: Measurement of specific IgG antibodies can indicate past or ongoing infections. For instance, detecting IgG antibodies against pathogens like Cytomegalovirus (CMV), Rubella, or Epstein-Barr virus (EBV) helps confirm prior exposure and immunity status.

Autoimmune Disorders: Elevated levels of specific IgG autoantibodies are associated with autoimmune diseases. For example, anti-cyclic citrullinated peptide (CCP) IgG antibodies are used as biomarkers for diagnosing rheumatoid arthritis.

Allergies: IgG4 subclass analysis is sometimes used to identify allergen-specific responses, particularly in delayed-type hypersensitivity reactions.

Vaccine Efficacy

Post-Vaccination Response: Measuring specific IgG titers after vaccination helps determine the level of immunity conferred by the vaccine. This is particularly important for vaccines against infectious diseases like hepatitis B, measles, and COVID-19.

Vaccine Development: In clinical trials, IgG analysis is used to assess the immunogenicity of new vaccines, providing data on antibody production and long-term immunity.

References

1. Aryal, S. "Immunoglobulin G (IgG)-Structure, Subclasses and Functions." (2018).
2. Murphy, et al. "Technology advancements in antibody purification." Antibody Technology Journal (2016): 17-32.

For research use only, not intended for any clinical use.